We have directly monitored the kinetics of fusion of intact HIV and SIV virions with target cells by photosensitized labeling of virus-resident membrane proteins which have become part of the host cell. Due to the stochastic nature of the CD4 and coreceptor-induced triggering events leading to HIV-1 Env-mediated fusion the process is slow; the time required to reaching half-maximal fusion is about 20 min. We have dissected these steps kinetically and analyzed the molecular features of the kinetic intermediates. Kinetic studies with HIV-1 Env-expressing cells using inhibitors indicate that the HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4. Our results suggest that the triggering events lead to destabilization of the membranes bearing HIV-1 Env, presumably as a result of self-insertion of the fusion peptide. We show that membrane microdomains or lipid rafts, which are specialized regions of host cell membrane enriched in glycosphingolipids, sphingomyelin and cholesterol, may play a role in organizing the viral envelope, CD4 and the appropriate chemokine receptor into a membrane fusion complex. Their role becomes crucial in primary lymphocytes, whose coreceptor levels are generally low. This process may also involve membrane signaling. The fusion reaction can be reversibly arrested at 37 0C by removal of cholesterol or glycosphingolipids from target cells, or by treatment with cytochalasin B. CXCR4 engagement and six-helix bundle formation only occurs after the release of the cytochalasin arrest. Our data indicate that a high degree of cooperativity is required to trigger the initial steps in HIV-1 Env-mediated fusion. Z01 BC 08303-28